In vivo NMR spectroscopy (MRS) is a powerful, non- invasive technique to study (human) brain metabolism. The combination of in vivo MRS and infusion of 13C-labeled substrates allows the quantitative measurement of metabolic fluxes, like cerebral glucose utilization and glutamate neurotransmitter cycling. However due to limitations in spectral and spatial resolution applications during functional activation have been limited. Here in vivo MRS methods at 4T will developed to overcome these problems and allow the routine study of functional metabolism. These methods will then be applied to study the relation between glucose metabolism and glutamatergic neuronal activity in the human brain during rest and visual stimulation. Short echo-time MRS will be improved through development of advanced methods for spectral quantification and multi-voxel localization incorporating prior knowledge on tissue composition (gray and white matter) and functional activity. Next, the technique will be applied to human brain in order to establish steady-state metabolite concentrations in various brain structures. The quantitative 1H MRS technique will be developed into a quantitative and dynamic (inverse) 1H-[13C]-MRS technique for the detection of 13C-labeled compounds with the high sensitivity of protons. The quantitative nature will be validated on an animal model by comparing the in vivo fractional enrichments of [4-13C]-glutamate and glutamine in rat brain, with those obtained in the extracts in vitro. The inverse MRS method will then be implemented to study the relationship between neuronal energy production and neurotransmitter activity and cycling by dynamically measuring the rates of glucose oxidation and glutamate neurotransmitter cycling. The results from this proposal will provide an essential, neurochemical link for the interpretation of functional brain mapping techniques, like fMRI and PET. A neurochemical basis of human brain mapping can provide valuable information about functional data comparisons between normal and diseased cerebral tissue.